Insulin/IGF-1 signalling (IIS) profoundly influences stress resistance and longevity in diverse organisms, possibly including humans. It is well known that IIS promotes aging by inhibiting DAF-16/FOXO proteins, but our lab has shown in C. elegans that IIS also directly inhibits the transcription factor SKN-1, which has conserved functions in stress resistance. SKN-1 promotes longevity, and contributes to stress resistance and longevity increases that are associated with reduced IIS. Our expression profiling shows that under normal conditions SKN-1 upregulates genes involved in many stress defense and cellular repair processes, and may directly repress numerous genes that limit stress resistance or longevity, including some IIS genes. In contrast, SKN-1 responds to stress by inducing a smaller group of detoxification genes. These findings show that SKN-1 plays a critical role in fundamental mechanisms that promote longevity, making it of major significance to understand how it functions and is regulated by IIS, and affects the organism when IIS is reduced. This project will investigate how IIS regulates gene expression by inhibiting SKN-1, and how SKN-1 promotes longevity and stress resistance in response to IIS reductions. Microarray-based profiling will be used to identify genes that are either up- or down-regulated by SKN-1 in response to reduced IIS, and to dissect effects of SKN-1 and DAF-16, thereby revealing processes and regulatory mechanisms that are controlled by both SKN- 1 and IIS. Our robust capability to analyze SKN-1 DNA binding in vivo by chromatin immunoprecipitation will be used to identify and compare genes that are directly regulated by SKN-1 under normal and IIS conditions. The functions of representative SKN-1 target genes will then be examined, work that will reveal processes through which SKN-1 contributes to these IIS phenotypes, and is likely to identify new longevity genes. Additionally, it will be investigated whether SKN-1 contributes to IIS stress and longevity phenotypes by acting only in the intestine, the major detoxification organ, or also in other tissues that are influenced by IIS. It will also be determined conclusively whether SKN-1 directly represses longevity-regulatory genes, and whether its repressive activity is relieved by stress. Finally, the regulatory effects of IIS on SKN-1 will be studied, first through an analysis of the 14-3-3 proteins PAR-5 and FTT-2. These proteins are likely effectors of IIS signals but regulate SKN-1 oppositely, with PAR-5 inhibiting SKN-1 and FTT-2 being a required cofactor. It will be investigated whether these 14-3-3 proteins interact with SKN-1 in response to IIS, and how FTT-2 might promote SKN-1 nuclear functions. In addition, proteomic approaches with which we are experienced will be used to identify proteins that associate with SKN-1 under normal and reduced IIS conditions, and may regulate its activities. PUBLIC HEALTH RELEVANCE: An understanding of mechanisms that increase productive lifespan and protect against chronic disease may provide the greatest cost/benefit ratio of any area of biomedical research. Reductions in insulin signalling promote longevity and resistance to metabolic stresses in diverse organisms. This project will use a powerful model organism, the nematode worm C. elegans, to investigate how a master regulator of mechanisms that defend against cellular damage and toxins contributes to the beneficial effects of reducing insulin signalling, and is itself controlled by the insulin signalling pathway.